The present invention generally relates to a method and apparatus for measuring the velocity of an object, and more specifically, to sensing light from an object with a light sensitive detector, the amplitude of that light signal having been modulated by an optical grating, and measuring the velocity of the object by analysis of the modulated light signal.
Cells and cell groupings are three-dimensional (3D) objects containing rich spatial information. The distribution of a tremendous variety of bio-molecules can be identified within a cell using an ever-increasing number of probes. In the post-genome era there is mounting interest in understanding the cell, not only as a static structure, but as a dynamic combination of numerous interacting feedback control systems. This understanding can lead to new drugs, better diagnostics, more effective therapies, and better health care management strategies. However, this understanding will require the ability to extract a far greater amount of information from cells than is currently possible.
The principal technologies for cellular analysis are automated microscopy and flow cytometry. The information generated by these mature technologies, although useful, is often not as detailed as desired. Automated microscopy allows two-dimensional (2D) imaging of from one to three colors of cells on slides. Typical video frame rates limit kinetic studies to time intervals of 30 ms.
Instruments known as flow cytometers currently provide vital information for clinical medicine and biomedical research by performing optical measurements on cells in liquid suspension. Whole blood, fractionated components of blood, suspensions of cells from biopsy specimens and from cell cultures, and suspensions of proteins and nucleic acid chains are some of the candidates suitable for analysis by flow cytometry. In flow cytometers specialized for routine blood sample analysis, cell type classification is performed by measuring the angular distribution of light scattered by the cells and the absorption of light by specially treated and stained cells. The approximate numbers of red blood cells, white blood cells of several types, and platelets are reported as the differential blood count. Some blood-related disorders can be detected as shifts in optical characteristics, as compared to baseline optical characteristics, such shifts being indicative of morphological and histochemical cell abnormalities. Flow cytometers have been adapted for use with fluorescent antibody probes, which attach themselves to specific protein targets, and for use with fluorescent nucleic acid probes, which bind to specific DNA and RNA base sequences by hybridization. Such probes find application in medicine for the detection and categorization of leukemia, for example, in biomedical research, and drug discovery. By employing such prior art techniques, flow cytometry can measure four to ten colors from living cells. However, such prior art flow cytometry offers little spatial resolution, and no ability to study a cell over time. There is clearly a motivation to address the limitations of existing cell analysis technologies with a novel platform for high speed, high sensitivity cell imaging.
A key issue that arises in cell analysis carried out with imaging systems is the measurement of the velocity of a cell or other object through the imaging system. In a conventional time-domain methodology, cell velocity is measured using time-of-flight (TOF). Two detectors are spaced a known distance apart and a clock measures the time it takes a cell to traverse the two detectors. The accuracy of a TOF measurement is enhanced by increasing detector spacing. However, this increases the likelihood that multiple cells will occupy the measurement region, requiring multiple timers to simultaneously track all cells in view. Initially, the region between the detectors is cleared before starting sample flow. As cells enter the measurement region, each entry signal is timed separately. The system is synchronized with the sample by noting the number of entry signals that occur before the first exit signal.
TOF velocity measurement systems are prone to desynchronization when the entry and exit signals are near threshold, noise is present, or expected waveform characteristics change due to the presence of different cell types and orientations. Desynchronization causes errors in velocity measurement which can lead to degraded signals and misdiagnosed cells until the desynchronized condition is detected and corrected. Resynchronization may require that all cells be cleared from the region between the detectors before restarting sample flow, causing the loss of sample.
Significant advancements in the art of flow cytometry are described in commonly assigned U.S. Pat. No. 6,249,341, issued on Jun. 19, 2001, and entitled IMAGING AND ANALYZING PARAMETERS OF SMALL MOVING OBJECTS SUCH AS CELLS, as well as in commonly assigned U.S. Pat. No. 6,211,955, issued on Apr. 3, 2001, also entitled IMAGING AND ANALYZING PARAMETERS OF SMALL MOVING OBJECTS SUCH AS CELLS. The specifications and drawings of each of these patents are hereby specifically incorporated herein by reference.
The inventions disclosed in the above noted patents perform high resolution, high-sensitivity 2D and 3D imaging using time delay integration (TDI) electronic image acquisition with cells in flow. These instruments are designed to expand the analysis of biological specimens in fluid suspensions beyond the limits of conventional flow cytometers. TDI sensors utilize solid-state photon detectors such as charge-coupled device (CCD) arrays and shift lines of photon-induced charge in synchronization with the flow of the specimen. The method allows a long exposure time to increase a signal-to-noise ratio (SNR) in the image while avoiding blurring. However, precise synchronization of the TDI detector timing with the motion of the moving targets is required. For example, if a target is to traverse 100 lines of a TDI sensor to build an image, and the blurring is expected to be less than a single line width, then the velocity of the target must be known to less than one percent of its actual value. It would thus be desirable to provide method and apparatus capable of producing highly accurate flow velocity for such moving targets.
Several methods have been suggested in the prior art to address the limitations of TOF velocity measurements, and to achieve highly accurate flow velocity for moving targets such as cells. One such technique is laser Doppler anemometry (LDA), in which one or more laser beams are used to interrogate a moving target. The Doppler frequency shift is detected as modulation from the interference of multiple beams that have traversed different paths in the apparatus. An example of such an apparatus is disclosed in U.S. Pat. No. 3,832,059, issued on Aug. 27, 1974, and entitled FLOW VELOCITY MEASURING ARRANGEMENT UTILIZING LASER DOPPLER PROBE. That apparatus employs two laser beams that are directed toward a moving target at converging angles. The back-scattered light from both beams is collected and focused on a common photodetector. Coherent interference at the detector generates modulation with frequency equal to twice the Doppler shift frequency, allowing the detection of the target velocity. While LDA is a functional technique, the LDA systems described in U.S. Pat. No. 3,832,059 are elaborate, expensive, and prone to instability.
In an attempt to overcome the problem of instabilities in the laser wavelength, an improved LDA apparatus is disclosed in U.S. Pat. No. 5,229,830, issued on Jul. 20, 1993, and entitled DOPPLER VELOCIMETER. In this improved apparatus, a rotating grating is added for the purpose of extracting diffraction side lobes for use in interrogating the target. This approach eliminates the wavelength dependency in the velocity measurement, but adds even more cost and complexity to the velocimeter.
An alternative approach to measuring object velocity by coherent light interference is found in LDA instruments that generate fringe patterns in the measurement field. Multiple laser beams create either stationary or propagating fringe patterns with known periods. Objects crossing the fringes modulate the light collected by the instruments. The modulation frequency is equal to the velocity divided by the fringe pattern period. LDA instruments of this type are disclosed in U.S. Pat. No. 4,148,585, issued on Apr. 10, 1979, and entitled THREE DIMENSIONAL LASER DOPPLER VELOCIMETER; and in U.S. Pat. No. 5,160,976, issued on Nov. 3, 1992, and entitled OPTICAL DETERMINATION OF VELOCITY USING CROSSED INTERFERENCE FRINGE PATTERNS. The apparatus disclosed in the first of these two patents uses a rotating diffraction grating and a beam splitter to generate four interrogation beams, which are then crossed in various combinations to measure velocity as a 3D vector. The apparatus disclosed in U.S. Pat. No. 5,160,976 uses specially constructed optical fiber bundles and two lasers of differing wavelength to generate two fringe patterns at the target, such that the patterns are oriented perpendicular to one another, in order to measure velocity as a 2D vector. Such fringe pattern velocimeters have been adapted for use in mapping complex flow fields. However, the cost, complexity, and instability inherent in delivering multiple coherent beams to the sample volume, with tightly constrained wavelengths and alignments, is a deterrent to their use. It would be desirable to provide an alternative method and apparatus to measure the velocity of an object entrained in a flow of fluid which is less costly, and more forgiving than these prior solutions to this problem.
The LDA systems described above rely on the mechanisms of Doppler shift and wave interference for the measurement of velocity. An alternative approach to measuring the velocity of objects in fluids is to collect data in short bursts at carefully timed intervals, i.e., to collect stroboscopic snapshots of the objects. This approach, which is related to but more complicated than the TOF method described above, is called object imaging velocimetry (PIV). One such system is disclosed in U.S. Pat. No. 4,729,109, issued on Mar. 1, 1988, and entitled METHOD AND APPARATUS FOR MEASURING THE DISPLACEMENTS OF PARTICLE IMAGES FOR MULTIPLE EXPOSURE VELOCIMETRY. In this patent, a strobe light source illuminates the target field. Cylindrical lenses collapse the image of the field into two orthogonal linear projections which are captured by linear arrays of photodetectors. Signals are collected from the detector arrays for every flash of the strobe light. For each axis, the signal from a first exposure is correlated with the signal from the subsequent exposure to measure target displacement. The displacement is converted to velocity by dividing the displacement by the time between exposures. The apparatus forms images in which contrast is created by light absorption by the target.
A PIV instrument utilizing target fluorescence is disclosed in U.S. Pat. No. 5,333,044, issued on Jul. 26, 1994, and entitled FLUORESCENT IMAGE TRACKING VELOCIMETER. In the apparatus described in this patent, a planar region in the field of flow is illuminated using a strobe-coherent light source. A detector forms 2D images of the fluorescent emission of objects stained with a fluorescent dye. Computer analysis of the sequence of captured images is used to track the motion of objects in the plane of illumination.
It should be noted that the stabilization and alignment of PIV systems are less problematic than in LDA systems, but the PIV systems require pulsed illumination or gated data acquisition to establish timing. The PIV systems also require arrays of detectors and elaborate data analysis to yield velocity measurements.
A technology that provides a simple, cost-effective alternative to LDA and PIV for measuring object velocity in fluids is based on the insertion of a grating with alternating opaque and transparent parallel bars in the light path of the photosensor. Light from moving objects is modulated by the optical grating pattern to create a signal with frequency directly proportional to the component of velocity perpendicular to the optical grating bars. If object motion is constrained to this perpendicular direction, then the frequency is equal to the true velocity divided by the period, or pitch, of the optical grating. A laser velocimeter based on this principle for measuring the velocity of a reflective surface moving relative to the instrument is disclosed in U.S. Pat. No. 3,432,237, issued on Mar. 11, 1969, and entitled VELOCITY MEASURING DEVICE. In the disclosed system of this patent, the target surface is illuminated with a continuous wave laser and light scattered by the moving surface is collected by a lens and then delivered to a photosensitive detector through a grating. The bars of the optical grating are oriented perpendicular to the axis of motion. An electronic frequency measuring circuit is used to determine the frequency of the photosensitive detector. The frequency is conveyed directly to a display device for viewing and conversion to velocity.
The application of this method to objects suspended in fluid is disclosed in U.S. Pat. No. 3,953,126, issued on Apr. 27, 1976, and entitled OPTICAL CONVOLUTION VELOCIMETER. In the disclosed apparatus of this patent, light collimated by a lens passes through the flow of fluid and is reflected by a mirror with alternating bars of reflective and absorptive material. The reflective bars return light through the flow of fluid to be collected by the lens. The lens focuses the reflected light on a photosensitive detector. An electronic circuit is used to estimate the frequency of the detector signal and to deliver the frequency to a display device for viewing.
It should be noted that the hardware signal processors used in early implementations of laser velocimeters have largely been displaced by computation-based digital signal processors. The demands on the photosensor signal processors vary with the nature of the application, but the most stringent applications demand high speed and high accuracy, under conditions of low SNR and rapidly varying flow velocity.
An example of an effective method for extracting velocity from the photosensor signal of a grating-based laser velocimeter is disclosed in U.S. Pat. No. 5,859,694, issued on Jan. 12, 1999, and entitled OPTICAL VELOCIMETER PROBE. In this patent, the digitized photosensor signal is captured in blocks of samples for processing. For each block, the signal processor executes the steps of generating a complex signal using the Hilbert transform, autocorrelating the complex signal, and extracting the phase for each time sample of the autocorrelogram. The autocorrelation is performed using the steps of a complex Fourier transformation, squaring the magnitude of the spectrum, and then applying an inverse Fourier transformation. Finally, an optimization routine finds a best-fit velocity value for the phase samples. The method described in this patent has the advantage of building SNR and delivering accurate velocity estimates, given long signal segments. However, the method is computation intensive, limiting the rate at which the velocity estimate is updated.
It would be desirable to utilize the principal of modulation of light from moving objects by the insertion of a periodic grating into the detector path for the purpose of measuring object velocity, and to further employ improved signal processing, superior control system design, and/or a unique grating design to achieve high precision velocity measurements in an imaging flow cytometer.
The present invention is directed to a system and method for determining a velocity of an object. This method will preferably be employed in conjunction with an imaging or optical system that is adapted to determine one or more characteristics of an object from an image of the object or light from the object. There is relative movement between the object and the imaging system, and although it is contemplated that either, or both, the object and imaging system may be in motion, the object will preferably be in motion and the imaging system will be fixed. When used in conjunction with an imaging system that incorporates a TDI detector, the velocity determined according to the present invention is used to provide a clocking signal to synchronize the TDI detector with the movement of the image over the TDI detector.
It should be understood that while portions of the following description, and the claims that follow, refer to xe2x80x9can object,xe2x80x9d it is clearly contemplated that the present invention is intended to be used with a plurality of objects, and is particularly useful in connection with imaging a stream of objects. In at least one embodiment, the stream of objects comprises moving cells or cell clusters. Furthermore, while the examples cited herein primarily relate to measuring the velocity of objects such as cells flowing in a stream of liquid, the present invention applies as well to objects attached to a solid substrate moving relative to the imaging system and to particles or liquid droplets flowing in a stream of gas.
Fundamental to the present invention are the illumination of an object, and the detection of light from the object. For the purpose of velocity measurement, the detected light must carry information about the change of position of the object over time. The object may modify the illumination light by scattering, refraction, diffraction, or absorption so that its wavelength is the same as that of the light received by the detector. Alternatively, fluorescence or phosphorescence at the object caused by the illumination light may result in light of different wavelengths than the illumination light to be received at the detector. Furthermore, the object may emit light used to determine the velocity of the object, without requiring prior illumination of the object by another light source.
The basic velocity measuring system includes xe2x80x9ca sensitive volume,xe2x80x9d through which objects, preferably entrained in a fluid, are illuminated. Light from the objects is directed along a collection path. The collected light is first converted into an electrical signal, and then the electrical signal is digitized. The digitized signal is analyzed to extract the velocity of an object. Thus, the velocity measuring system will preferably include a light source that illuminates the objects in motion (unless the object self-emit light without any need for illumination by a light source), a light sensitive detector for receiving light from the objects, and electronic components to manipulate the signal from the light sensitive detector to determine a velocity. The light source may be a laser, a light emitting diode, a filament lamp, or a gas discharge arc lamp, for example, combined with optical conditioning elements such as lenses, apertures, and filters to deliver the desired wavelength(s) of light to the object at an intensity required for determination of the velocity of the object. The detector may be a photomultiplier tube or a solid-state photodetector and may be combined with optical conditioning elements such as lenses, apertures, and filters to deliver the desired wavelengths of light from the object to the detector.
Light from the object is received as the object passes through the sensitive volume. Because the sensitive volume is bounded by a profile of an illumination field and by an acceptance window of the light sensitive detector, it would be possible to estimate the velocity of the object from the time required for the object to pass through the sensitive volume. However, the sensitive volume is bounded by gradients rather than distinct edges, and it may be impractical to maintain the dimensions of the sensitive volume with a high degree of accuracy.
To address the above-noted problem, one or more optical gratings is used to establish a distance measurement scale for computing velocity, eliminating the concern for maintaining the dimensions of the sensitive volume with a high degree of accuracy. The optical gratings are preferably fabricated using high precision methods, such as photolithographic etching, to create patterns of alternating bars of opaque and transparent material. The accuracy required for the fabrication of the optical gratings is a function of the accuracy required in the velocity measurement. Preferably the optical grating includes alternating sequences of opaque strips and transparent strips of substantially equal width.
As noted above, when the velocity measurement system is used in conjunction with an imaging system that preferably employs a TDI detector. In TDI imaging, the charge in a TDI detector is moved in synchrony with an image that is incident on the TDI detector, enabling a thousand-fold increase in integration times over conventional frame imaging. TDI imaging is conventionally performed by clocking a pixel output from the detector using an encoder that directly measures sample movement, keeping the charge on the detector synchronized with the image. Since the transport of objects by a fluid stream prevents the use of an encoder, systems imaging objects in flow require an alternative means of synchronizing the TDI detector with the moving object and thus, with the moving image of the object across the TDI detector. The present invention provides an acceptable approach for measuring the velocity of moving objects (such as cells) for use in such TDI based imaging systems.
The present invention encompasses two different configurations of optical gratings, and two different signal processing methods, which are used to analyze the modulated signal produced by a detector in response to the modulated light produced by an optical grating, thereby enabling the velocity of an object to be determined. Preferably, each signal processing embodiment is executed under the control of a supervisory program appropriate to the particular method employed.
In one embodiment, the signal is homodyned to a baseband and the passband is limited to improve the SNR. The frequency of the modulated light is extracted in the time domain from signal packets selected for low phase interference. This method is well suited for execution in a fast pipeline processor and delivers relatively high accuracy and relatively high sensitivity. Another embodiment employs two optical gratings displaced from one another along the flow axis to modulate light from the object, so that the modulated light is incident on two different detectors. Cross-correlation of the two signals from the detectors rapidly delivers a highly accurate estimate of velocity of the object. The performance of these two embodiments is preferably optimized by the inclusion of supervisor programs, which rapidly adapt to changing flow velocity and varying signal strength from the detector(s). The choice of the embodiment that is employed will depend on the particular demands of an application for determining the velocity of an object.
More specifically, a velocity measurement system in accord with the present invention includes an optical element disposed so that light traveling from an object passing through a sensitive volume is directed by an optical element along a collection path. At least one optical grating is disposed in the collection path and modulates the light, producing modulated light having a modulation frequency corresponding to a velocity of the object. At least one light sensitive detector is disposed in the collection path and converts the modulated light into an electrical signal. The velocity measurement system also includes means for converting the electrical signal into a sequence of digital samples, and means for processing the sequence of digital samples to determine a velocity of the object corresponding to the electrical signal.
Preferably, the means for converting the electrical signal into the sequence of digital samples comprises at least one analog-to-digital converter. It is likely that most such systems will include at least one amplifier electrically coupled each light sensitive detector, for amplifying the electrical signal before conversion of the electrical signal into a sequence of digital samples. In several embodiments, bandpass filters are employed to filter the electrical signal before conversion into a sequence of digital samples. Alternatively, the electrical signal may be converted into a sequence of digital samples immediately after amplification, and a digital bypass filter may be utilized. The means for processing the sequence of digital samples can comprise a computer, an application specific integrated circuit (ASIC), or a digital oscilloscope.
Preferably, a velocity measurement system in accord with the present invention will further include a system controller for controlling the acquisition of the electrical signal, for controlling the means for converting the electrical signal into a sequence of digital samples, and for controlling the means for processing the sequence of digital samples. The system controller is preferably a computer or other programmed computing device, however, it should be understood that an ASIC can also be beneficially employed as a system controller. Preferably the system controller will regulate a gain of an amplifier that amplifies the electrical signal, regulate a threshold applied to frequency measurements so that measurements made under the condition of inadequate SNR are rejected, and/or regulate a frequency of a pair of baseband converter local oscillators in response to variations in a velocity of the object.
The light from an object passing through the sensitive volume can include light scattered by that object, an unstimulated emission from the object, or a stimulated emission from the object. While it is contemplated that the velocity measuring system and method of the present invention can use ambient light, at least one light source is preferably incorporated into such a system for illuminating the sensitive volume (unless the object self-emits light without requiring prior illumination from another source). Such a light source can also stimulate a fluorescent emission from an object passing through the sensitive volume. Incident light can be at least partially absorbed by an object passing through the sensitive volume, so that the light directed by the optical element will have been affected by the absorption of light by the object. Alternatively, incident light is reflected from an object passing through the sensitive volume toward the optical element. The light source can be one or more of a coherent light source, a non-coherent light source, a pulsed light source, a continuous light source, a continuous wave laser, a pulsed laser, a continuous wave incandescent lamp, a strobe arc lamp, and may include an optical filter for selecting a limited spectrum for illumination.
Velocity measurement systems in accord with the present invention preferably enable a flow of fluid, in which objects are entrained, to pass through the sensitive volume, such that a velocity of an object so entrained can be measured. In other embodiments, velocity measurement systems in accord with the present invention enable a support, on which objects are disposed, to pass through the sensitive volume, such that a velocity of the objects are disposed, to pass through the sensitive volume can be measured. At least one embodiment will comprise a stage-based motion system with a high-resolution linear encoder.
It is anticipated that velocity measuring systems, or combined velocity measurement and imaging systems can beneficially include a mechanism for sorting objects disposed downstream of the sensitive volume(s).
In one embodiment in which the optical grating has a uniform pitch, the means for converting the electrical signal into a sequence of digital samples includes an amplifier that produces an amplified electrical signal based on the electrical signal received from the detector. The means also includes a bandpass filter for filtering the amplified electrical signal to produce a passband signal. The means further includes a baseband converter for converting the passband signal into a pair of signals that represent the passband signal with two components, a real component and an imaginary component. Each of the two baseband signals is converted to a sequence of digital samples by an analog-to-digital converter.
For embodiments wherein the at least one optical grating has a uniform pitch, the means for processing the sequence of digital samples preferably determines the velocity from a first derivative of a time series of phase samples of the electrical signal, after calculating those phase samples from a baseband complex representation of the electrical signal. This processing of the electrical signal can be executed by a programmed computer, an ASIC, other hardwire logic circuits, or a digital oscilloscope.
In another embodiment, two substantially identical optical gratings, each having a non-uniform pattern of opaque bar and transparent gap widths, are disposed along an axis of motion of an object such that the object sequentially traverses the sections.
In one preferred embodiment that incorporates non uniform optical gratings, a beam splitter is disposed in the collection path, such that a portion of the light traveling from an object along the collection path is diverted along a second collection path. A second non uniform optical grating is disposed in the second collection path, such that the second optical grating also modulates the light, producing modulated light have a modulation frequency corresponding to a velocity of an object passing through the sensitive volume. A second light sensitive detector is disposed in the second collection path, and converts the light modulated by the second optical grating into an electrical signal.
In an embodiment incorporating two optical gratings with a non uniform pitch, the means for converting the electrical signal into a sequence of digital samples includes a first amplifier coupled to the detector that is disposed in the collection path. The first amplifier amplifies the electrical signal from a first light sensitive detector, producing an amplified electrical, and a first bandpass filter filters the amplified electrical signal to produce a first passband signal. A first analog-to-digital converter converts the passband signal to a first sequence of digital samples. The means for converting the electrical signal into a sequence of digital samples also includes a second amplifier disposed in a second collection path. The second amplifier amplifies the second electrical signal, producing a second amplified electrical signal that is filtered by a second bandpass filter, producing a second passband signal. The second passband signal is input to a second analog-to-digital converter, which converts the second passband signal to a second sequence of digital samples. Alternatively, the bandpass filter operation can be accomplished using digital bandpass filters following the analog-to-digital converters.
Preferably when two non uniform optical gratings are employed, each optical grating is aligned in series along an axis of motion corresponding to an object that the light is collected from, and the means for processing the sequence of digital samples to determine a velocity of an object calculates an amplitude peak of a cross-correlogram generated by a convolution of the electrical signal from the first light sensitive detector disposed in the collection path and the second electrical signal from the second light sensitive detector disposed in the second collection path.
Embodiments with optical gratings of non uniform pitch also preferably include a control system controllably connected to the means for converting the electrical signal into a sequence of digital samples and the means for processing the sequence of digital samples. The control system preferably regulates the gain of each amplifier in response to varying electrical signal levels, and regulates an upper and a lower limit for time shifting the cross-correlation step in response to variations in the velocity of an object.
As noted above, velocity measurement systems in accord with the present invention can be incorporated into imaging systems that also determine non-velocity characteristics of an object passing through the sensitive volume, by employing at least one TDI detector that using the velocity for a clocking function. Such combination velocity measurement and imaging systems preferably include a second optical element disposed so that light traveling from an object passing through a second sensitive volume passes through the second optical element and travels along yet another collection path, and at least one additional detector that determines a non-velocity related characteristic of an object passing through the second sensitive volume. Preferably, the second sensitive volume, the second optical element, and the at least one additional detector are disposed downstream from the sensitive volume, optical element, and light sensitive detector of the velocity measuring portion of the combined system.
It is anticipated that when the objects in question are cells, the accurate determination of cell velocity for use in a TDI-based imaging system for cell analysis, in accord with the present invention, will greatly expand the amount of information that can be extracted from cells. It is expected that the present invention will enable systems to process sample sizes of about 100 million cells, and analyze the samples with high spectral and spatial resolution, at very high speeds. Such a system will have clinical applications that include non-invasive prenatal diagnosis via maternal peripheral blood, routine cancer screening and eventually, therapeutic applications involving the isolation, modification and re-implantation of rare cells. In addition, the present invention is directed to a general purpose velocity detection method that can be applied to other applications where an accurate determination of the velocity of an object or the velocity of a flow is required.